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. 2024 Jan 23;96(3):1214-1222.
doi: 10.1021/acs.analchem.3c04406. Epub 2024 Jan 8.

Quantification of mRNA in Lipid Nanoparticles Using Mass Spectrometry

Mark S Lowenthal  1 , Abigail S Antonishek  1 , Karen W Phinney  1
Affiliations

Quantification of mRNA in Lipid Nanoparticles Using Mass Spectrometry

Mark S Lowenthal et al. Anal Chem. .

Abstract

Lipid nanoparticle-encapsulated mRNA (LNP-mRNA) holds great promise as a novel modality for treating a broad range of diseases. The ability to quantify mRNA accurately in therapeutic products helps to ensure consistency and safety. Here, we consider a central aspect of accuracy, measurement traceability, which establishes trueness in quantity. In this study, LNP-mRNA is measured in situ using a novel liquid chromatography-mass spectrometry (LC-MS) approach with traceable quantification. Previous works established that oligonucleotide quantification is possible through the accounting of an oligomer's fundamental nucleobases, with traceability established through common nucleobase calibrators. This sample preparation does not require mRNA extraction, detergents, or enzymes and can be achieved through direct acid hydrolysis of an LNP-mRNA product prior to an isotope dilution strategy. This results in an accurate quantitative analysis of mRNA, independent of time or place. Acid hydrolysis LC-MS is demonstrated to be amenable to measuring mRNA as both an active substance or a formulated mRNA drug product.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
LC-MS/MS (MRM) TICs of acid hydrolyzed calibrators (pure nucleobases) and QCs (naked mRNA).
Figure 2
Figure 2
LC-MS/MS (MRM) XICs of acid hydrolyzed LNP-mRNA (ProMab).
Figure 3
Figure 3
Residual protein impurity detected in LNP-mRNA products.
See this image and copyright information in PMC

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References

    1. Crank M. C.; Ruckwardt T. J.; Chen M.; Morabito K. M.; Phung E.; Costner P. J.; Holman L. A.; Hickman S. P.; Berkowitz N. M.; Gordon I. J.; Yamshchikov G. V.; Gaudinski M. R.; Kumar A.; Chang L. A.; Moin S. M.; Hill J. P.; DiPiazza A. T.; Schwartz R. M.; Kueltzo L.; Cooper J. W.; Chen P.; Stein J. A.; Carlton K.; Gall J. G.; Nason M. C.; Kwong P. D.; Chen G. L.; Mascola J. R.; McLellan J. S.; Ledgerwood J. E.; Graham B. S. A proof of concept for structure-based vaccine design targeting RSV in humans. Science 2019, 365 (6452), 505–509. 10.1126/science.aav9033. - DOI - PubMed
    1. Rohner E.; Yang R.; Foo K. S.; Goedel A.; Chien K. R. Unlocking the promise of mRNA therapeutics. Nat. Biotechnol. 2022, 40 (11), 1586–1600. 10.1038/s41587-022-01491-z. - DOI - PubMed
    1. Polack F. P.; Thomas S. J.; Kitchin N.; Absalon J.; Gurtman A.; Lockhart S.; Perez J. L.; Pérez Marc G.; Moreira E. D.; Zerbini C.; Bailey R.; Swanson K. A.; Roychoudhury S.; Koury K.; Li P.; Kalina W. V.; Cooper D.; Frenck R. W. Jr.; Hammitt L. L.; Türeci Ö.; Nell H.; Schaefer A.; Ünal S.; Tresnan D. B.; Mather S.; Dormitzer P. R.; Şahin U.; Jansen K. U.; Gruber W. C. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J. Med. 2020, 383 (27), 2603–2615. 10.1056/NEJMoa2034577. - DOI - PMC - PubMed
    1. Medicine N. L. o. ClinicalTrials.gov.
    1. Whitley J.; Zwolinski C.; Denis C.; Maughan M.; Hayles L.; Clarke D.; Snare M.; Liao H.; Chiou S.; Marmura T.; Zoeller H.; Hudson B.; Peart J.; Johnson M.; Karlsson A.; Wang Y.; Nagle C.; Harris C.; Tonkin D.; Fraser S.; Capiz L.; Zeno C. L.; Meli Y.; Martik D.; Ozaki D. A.; Caparoni A.; Dickens J. E.; Weissman D.; Saunders K. O.; Haynes B. F.; Sempowski G. D.; Denny T. N.; Johnson M. R. Development of mRNA manufacturing for vaccines and therapeutics: mRNA platform requirements and development of a scalable production process to support early phase clinical trials. Transl Res. 2022, 242, 38–55. 10.1016/j.trsl.2021.11.009. - DOI - PMC - PubMed